An essential domain within Cdc34p is required for binding to a complex containing Cdc4p and Cdc53p in Saccharomyces cerevisiae.
The CDC34 gene of the yeast Saccharomyces cerevisiae encodes a ubiquitin-conjugating protein that transfers ubiquitin onto substrates to signal rapid degradation via the proteasome. Cdc34p has been implicated in signaling the destruction of a variety of substrates including the cyclin-dependent kinase inhibitor, Sic1p, which must be degraded for cells to enter S-phase. Mutants lacking CDC34 activity fail to degrade Sic1p and fail to enter S-phase, a phenotype that is also shared with cells lacking CDC4 and CDC53 activity. Here we demonstrate that Cdc4p, Cdc34p, and Cdc53p interact in vivo. We have mapped a Cdc4p/Cdc53p-binding region on Cdc34p; this region is essential for S-phase entry and thus the association of these three proteins is required for Sic1p degradation. All three proteins migrate in gel filtration to sizes that greatly exceed their actual size suggesting that they form stable associations with other proteins and we observe Cdc4p, Cdc34p, and Cdc53p fractionating into overlapping families of high molecular weight complexes. Finally, we demonstrate that Cdc4p, Cdc34p, and Cdc53p are stable throughout the cell cycle and that Cdc34p permanently resides as part of a complex throughout the cell cycle. This suggests that all Cdc34p substrates are ubiquitinated by a similar high molecular weight complex.
Mathias
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Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and the Walther Oncology Center, Indianapolis, Indiana, 46202-5122, USA. neal@biochem4.iupui.edu
Steussy
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Goebl
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D064173:Anaphase-Promoting Complex-Cyclosome; D001665:Binding Sites; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D044842:Cullin Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D003546:Cysteine Endopeptidases / Q000378:metabolism; D044783:F-Box Proteins; D005656:Fungal Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction / Q000502:physiology; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
C080443:CDC4 protein, S cerevisiae; C100988:Cdc53 protein, S cerevisiae; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D044783:F-Box Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C577449:CDC34 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases